Removal of agglomerates

ABSTRACT

The present disclosure provides a method of dispersing agglomerated material in a preparation comprising influenza proteins. The method comprises subjecting the preparation to sonication.

CROSS REFERENCE TO RELATED APPLICATIONS

The present applications claims priority from Australian Application No.2018902497 filed on 10 Jul. 2018 and entitled “Removal of agglomerates”.The entire contents of that application are hereby incorporated byreference.

FIELD

The present disclosure relates to a method of dispersing agglomerates ina preparation comprising influenza antigens and in particular, the useof this method in the production of influenza vaccines.

BACKGROUND

Influenza vaccines are considered the most effective method to preventinfection. The first influenza vaccines were whole virus preparations[1]. The current manufacturing process of inactivated trivalent andquadravalent influenza vaccines (TIV and QIV respectively) is based uponchemical disruption or “splitting” of the influenza virus, which wasintroduced in the 1960s [2]. Chemical disruption (by detergent orsolvent) was found to reduce the reactogenicity of the vaccine without,in many cases, compromising the immunogenicity. Due to the highvolatility of solvents, all commercially available influenza vaccinesare disrupted or split with detergent. However, the concentration ofdetergent utilized to disrupt the whole virion exceeds acceptable limitswithin the vaccine and therefore must be removed to permissible levels.

The consequence of removal of the detergent is that the resulting splitvirions develop agglomerates or agglomerated material. The developmentof agglomerates relates to the strain and the level/type of detergentutilized in the splitting process. In many cases vaccines and otherpharmaceutical products contain either residual detergent or anadditional detergent/chemical to maintain appropriate quality attributesof the vaccine. However, it has long been established that the presenceof detergent, in particular in the context of vaccination, rendersantigens more soluble potentiating a decrease in the immunogenicity andhence effectiveness of the vaccine.

SUMMARY

The present disclosure provides a method of dispersing agglomeratedmaterial in a preparation comprising influenza proteins or virus, themethod comprising subjecting the preparation to sonication.

The present disclosure also provides a method of producing an influenzavaccine the method comprising producing a preparation comprisinginactivated or split influenza virions and sonicating the preparation.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Repeatability of optical density turbidity (ODT) assay forinfluenza virus vaccine (IVV) IVV Drug Matrix of strains ofA/Victoria/361/2011 (H3N2), A/California/07/2009 (H1N1) andB/Hubei-Wujiagang/158/158/2009 (B Yamagata).

FIG. 2: Influence of sonication with respect to the amount of energydelivered (Joules/mL) and heat (37° C., 30 min) on dispersion of H3N2A/Victoria/361/2011 IVV Drug Matrix, by ODT analysis.

FIG. 3: Influence of sonication with respect to intensity of energyinput (amplitude) on dispersion of H3N2 A/Victoria/361/2011 IVV DrugMatrix, by ODT analysis.

FIG. 4: ODT results demonstrating sonication effectively dispersesagglomerates and maintains the dispersed state of H3N2A/Victoria/210/2009 IVV Drug Matrix over time, compared with untreatedand PS80-treated samples.

FIG. 5: Average intensity particles size distribution (PSDs) (n=5) of(A) untreated, (B) PS80-treated and (C) sonicated H3N2 IVV Drug Matrixof A/Victoria/210/2009, analyzed by DLS over 24 weeks.

FIG. 6: Single radial immunodiffusion (SRID) analysis of untreated,detergent (PS80)-treated and sonicated IVV Drug Matrix (H3N2;ANictoria/210/2009) over 24 weeks.

FIG. 7: EM micrographs representing IVV Drug Matrix (MPH), IVV DrugMatrix post sonication (Sonicated MPH) and IVV Drug Matrix in thepresence of polysorbate 80 (MPH+PS80). All samples were analyzed by EMat 0, 1, 2 and 6 month time points.

FIG. 8: ODT results for four seasonal strains (A/Victoria/361/2011(H3N2), A/California/7/2009 (H1N1), B/Hubei-Wujiagang/158/2009 (BYamagata) and B/Brisbane/60/2008 (B Victoria)) of influenza, pre- andpost-sonication treatment (Son) at time 0, 3 and 6 months.

FIG. 9: Average particle size distributions (PSDs) (n=5) of untreated(left) and sonicated (right) IVV Drug Matrix material forA/Victoria/361/2011 (A, B), A/California/7/2009 (C, D),B/Hubei-Wujiagang/158/2009 (E, F) and B/Brisbane/60/2008 (G, H),analyzed by DLS over six months.

FIG. 10: Sonication exposure time (min) over IVV Drug Matrix batchvolume (MPH volume mL) corresponding to ODT≥80%, for various IVV DrugMatrix batch volumes of 60, 500 and 1000 ml.

FIG. 11: Sonication energy input over processing time corresponding toODT≥80%, for various IVV Drug Matrix batch volumes of 60, 500 and 1000ml.

FIG. 12: Sonication energy input required to achieve ODT≥80%, using aflow-through device versus batch volume of IVV Drug Matrix (60, 500 and1000 ml).

FIG. 13: Linear relationship between the amount of agglomeration in IVVDrug Matrix (depicted as % ODT) and the predicted number ofglycosylation sites present on the HA molecule.

DETAILED DESCRIPTION

The present disclosure describes the use of a sonication method toeffectively disperse agglomerated material in influenza virus vaccine(IVV) drug substance or IVV drug product and will be referred to hereafter as IVV Drug Matrix. The feasibility of sonication was assessed onthe H3N2 strain of influenza virus since this sub-strain of influenza Aexhibits the greatest level of agglomeration compared to non-H3N2strains and B influenza.

One example of the disclosure provides a previously unseen approachsince inhibition of protein agglomeration is commonly achieved by theaddition of a compatible excipient(s) to the formulation. For example,excipients such as sugars, polyols, amino acids, salts, polymers andsurfactants have been found to stabilize agglomerates by preferentialinteractions [(Arakawa et al (1991); Timasheff (1998)], increased rateof protein folding [Wang et al (1995); Frye and Royer (1997)], reductionof solvent accessibility and conformational mobility [Kendrick et al(1997)] as well as increased solvent viscosities [Jacob and Schmid(1999)].

The present disclosure avoids the need for these additives, which canadversely impact the immunogenicity of the vaccine and cause unwantedside effects in individuals receiving the vaccine.

In one example, the disclosure provides a method of dispersingagglomerated material in a preparation comprising influenza proteins,the method comprising subjecting the preparation to sonication.

In another example, the disclosure provides a method of producing aninfluenza vaccine the method comprising producing a preparationcomprising inactivated or split influenza virions and sonicating thepreparation.

Preparation for sonication can be whole virion, split virion, subunitvaccine or recombinant vaccine. In order to facilitate filtration forsterility of the preparation it is preferable that the percentage ofagglomerates is less than 10% in the final preparation.

In an example, the preparation comprises influenza haemagglutinin, forexample, the preparation comprises split influenza virions. In oneexample, the preparation is substantially free of detergent. As usedherein, the phrase “substantially free of detergent” means having alevel of less than 0.02%. For example, the level of detergent is lessthan 200 ppm. In a further example, the level of detergent is less than50 ppm.

Typically the sonication is conducted for a time and at intensity suchthat at least 50% of agglomerates present in the preparation aredispersed. The sonication energy produced for the disruption ofagglomerates can be delivered to the target IVV Drug Matrix by one ofthree ways: (1) Energy is transferred directly via the Sonicator probesuspended in the IVV Drug Matrix, (2) IVV Drug Matrix passes across thevibrating tip of the sonicator's probe tip enclosed in a flow-throughdevice or (3) Indirect energy transfer through metal or glass tubing towhere the IVV Drug Matrix passes through. All sonication methods requirethe transfer of at least 89 Joules/mL of energy to disperse at least 50%of the agglomerates within the IVV Drug Matrix.

The vaccine produced by the method of the present disclosure may be amonovalent seasonal vaccine or a monovalent pandemic vaccine. In anotherexample, the vaccine of the present disclosure is a multivalent vaccinesuch as trivalent and quadravalent vaccines.

The vaccine produced by the method of the present disclosure typicallycomprises influenza A and influenza B antigens and is, for example,substantially free of agglomerated material. In one example, the phrase“substantially free of agglomerated material” means that more than 50%(about 90 Joules/mL sonication) of the material is not aggregated. In afurther example, at least 60% (about 134 Joules/mL sonication), 70%(about 178 Joules/mL sonication) or 80% (about 223 Joules/mL sonication)of the material is not aggregated. In another example, at least 90%(about 267 Joules/mL sonication) of the material is not aggregated.

As is described below the method of the present disclosure provides anumber of unexpected advantages. Firstly, the method is highly efficientat dispersing agglomerates present in the influenza antigen preparation.Surprisingly these dispersed agglomerates do not re-aggregate despiteextended periods of storage (4° C.). Further it was shown that a vaccineproduced using the method of the present disclosure was able to elicit astronger immune response in the ferret model than vaccines comprisingthe same antigens but untreated or treated with additives to disperseagglomerates.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that prior publication (or information derived from it) orknown matter forms part of the common general knowledge in the field ofendeavor to which this specification relates.

All publications mentioned in this specification are herein incorporatedby reference in their entirety.

It must be noted that, as used in the present specification, thesingular forms “a”, “an” and “the” include plural forms unless thecontext clearly dictates otherwise. Thus, for example, reference to “anagent” includes a single agent, as well as two or more agents; referenceto “a molecule” includes a single molecule, as well as two or moremolecules; and so forth.

Examples Methods Sonication of IVV Drug Matrix Direct Probe SonicationMethod

IVV Drug Matrix was processed using a ‘direct’ sonication method,whereby the sonicator horn/probe is immersed directly into the samplecontained within a beaker.

A Branson Model 450 Sonifier® (Branson Ultrasonics) was used to performsonication and comprised four components, including a power unit, model102C convertor and a 0.5 inch tapped horn.

To assess the efficiency of the sonication process, a Bandelin SONOPULSSonicator (Bandelin Electronic GmbH & Co. KG) was used. The setupconsisted of a GM3200 Sonifier power unit, UW3200 convertor, SH213Gbooster and TT13 13 mm titanium tip. Sonication of IVV drug substancematerial was performed in a beaker as per the method described for theBranson Sonicator.

IVV Drug Matrix samples were prepared in 10 ml batches and placed in aclean 30 ml beaker, which was secured onto a clamp stand with theSonicator probe sufficiently immersed into the sample solution (i.e. thedistance between the Sonicator tip and the base of the beaker wasapproximately 1 mm). Sonication was then performed delivering a range ofenergy input while varying the rate of energy transfer (amplitude):

The range of sonication energy delivered was measured as Joules (energy)per milliliter of IVV Drug Matrix and included: 0 Joules/mL, 83Joules/mL, 165 Joules/mL, 259 Joules/mL, 345 Joules/mL.

To avoid overheating of the sample, sonication was conducted in at leasttwo parts and the beaker left to cool briefly on ice in betweenprocessing. Final sonicated IVV drug substance samples were transferredinto plastic tubes and stored at 2-8° C. prior to further analysis.

Flow-Through Sonication Method

To assess the scalability of the sonication process, a Bandelin SONOPULSSonicator (Bandelin Electronic GmbH & Co. KG) fitted with a flow-throughdevice was used. The setup consisted of a GM3200 Sonifier power unit,UW3200 convertor, SH213G booster, TT13 13 mm titanium tip and a DG 4 Gflow through processing vessel. Sonication of IVV drug substancematerial was circulated through the sonicator's flow-through device bymeans of a 520U peristaltic pump (Watson & Marlow, Australia).

Measurement of Agglomerated Material Optical Density Turbidity (ODT)Assay

To assess the degree of non-agglomerated material in the vaccineintermediate product IVV drug substance, the level of recovered proteinutilizing optical density (OD) at A280 nm of the supernatant followingthe application of a modest centrifugal force was determined (Tay et al:Investigation into alternate testing methodologies for characterizationof influenza vaccine. Human Vaccine Immunotherapy 2015 11 (7) 1673-84).Since the proportion of protein in the pellet after centrifugationdirectly correlates to the degree of agglomerated material within thesample, a higher recovery in the supernatant would correspond to agreater proportion of dispersed protein. This assay was termed theoptical density turbidity (ODT) method; protein recovery values rangefrom 0 to 100% (presented as % ODT herein), which increase with theamount of dispersed protein in the sample.

Several attributes of the assay were evaluated for three influenzastrains, including H1N1; A/California/07/2009, H3N2; A/Victoria/361/2011and B; B/Hubei Wujiagang/158/158/2009. Validation results demonstratedrepeatability (% CV) of 2.9, 3.1 and 3.1% (respectively), precisioninter-lot variability (% CV) of 8.8, 6.5 and 3.4% (respectively),intermediate precision of 0.4%, 6.3% and 0.2% (respectively),statistically insignificant differences between operators of p=0.81,0.13 and 0.78, (respectively), and expectable linearity between expectedand observed results (R=0.9685). Typical % ODT profiles for replicatelots of IVV drug substance representing seasonal vaccine sub-types:H1N1, H3N2 and Influenza B are detailed in FIG. 1.

Further this assay was correlated with alternative methods ofagglomeration characterization, including dynamic light scattering (DLS)and asymmetric field flow fractionation (A4F). The low level ofvariation from ODT analysis indicated that this assay was highlysuitable for the assessment of agglomeration in intermediate vaccinematerial.

Dynamic Light Scattering (DLS)

Particle size analysis by DLS was used as a complementary method to theODT assay to further understand the agglomeration characteristics withinthe samples. The DLS technique is based on the measurement of theBrownian motion of proteins in solution, which is the random movement ofparticles due to collisions with the surrounding solvent molecules. TheBrownian motion induces time dependent fluctuations in the intensity ofscattered light which is measured by DLS and generates a particle sizedistribution (PSD) for the sample.

DLS measurements were performed using the Malvern Zetasizer Nano SeriesZS (Malvern Instruments Ltd). Several properties which influence theBrownian motion of particles in solution was pre-determined, includingthe density (DA-100M Density Meter, Mettler Toledo), viscosity (Lovis2000M Microviscometer, Anton Paar) and refractive index (30GSRefractometer, Mettler Toledo) of each sample analyzed. Pre-treatment ofsamples involved centrifugation for 1 minute at 8000 rpm to remove anyextraneous material or sediment containing large particles which areunstable and subsequently interfere with the analysis. The resultingsupernatant component of each sample was then withdrawn and assessed byDLS; each sample measurement was based on five replicates (n=5)performed at a backscatter angle of 173° and equilibrated to atemperature of 25° C. for three minutes.

Influenza Antigenicity Assessment Single Radial Immunodiffusion (SRID)

SRID assays were performed as previously described [Williams et al,1980]. Briefly, reference and test antigen material was diluted 1:1, 2:3and 1:3 in PBS-containing 1% Zwittergent solution (Calbiochem,Darmstadt, Germany), and added to duplicate wells of agarose gelscontaining polyclonal antiserum. Gels were incubated for 72 hrs inhumidified chambers, dried onto glass plates, and stained with Coomassiebrilliant blue R-250 (Sigma, Calif., USA). Circular zones ofantigen-antibody precipitation were measured, and HA concentration wascalculated by the parallel line bioassay method in comparison to IZPstandards (15), and test validity was confirmed using a ‘g’ test(g≤0.061) (16).

Electron Microscopy (EM) Imaging

Negative staining EM was performed by the agar diffusion filtrationmethod adopted by Hayat and Miller (1990). Three grids of each samplewere prepared; IVV drug substance was diluted 1 in 50 with phosphatebuffered saline (PBS; pH 7.2) to provide a discontinuous monolayer. Thesample (1 μL) was applied to a formvar-coated copper electron microscopegrid, which was then inverted onto a 2% w/v agar plate. Upon settling ofthe grids onto the agar plate (i.e. the liquid absorbed by the agar),the grid was floated on a drop of negative stain (2% w/v sodiumphosphotungstate at pH 7.0). After twenty seconds, the grid was thenlifted and excess stain removed by contacting the edge of the grid witha small strip of torn Whatman No. 1 filter paper. The remaining thinfilm of stain was left to air-dry prior to examination by the electronmicroscope.

Results Disruption of Agglomerated IVV Drug Matrix by Sonication

The most effective method to disperse agglomerated material wasdetermined using a H3N2 sub-strain of influenza because of itspropensity to form the greatest level of agglomerated materialpost-detergent disruption. Several methods were assessed to establishwhether agglomerated material following detergent disruption could bedispersed.

The first approach involved using high frequency sound waves bysonication to disperse agglomerates within IVV drug substance materialof A/Victoria/361/2011, sub-type H3N2 (FIG. 2). A direct sonicationmethod was used in which localized and highly intense ultrasonic wavesof energy could be transmitted directly from the probe and into a beakercontaining the sample. The samples (prepared in 10 ml batches) weresubjected to a range of sonication input energy ranging from 83Joules/mL-345 Joules/mL and the extent of dispersion evaluated by theODT assay. The results demonstrate a linear relationship between theamount of energy input and the level of agglomerates disassociated, i.e.% ODT values ranged from 50% to 80% after sonication compared with 40%when untreated (FIG. 2). Interesting to note was that the level ofdispersion reached a maximum at 259 Joules/mL, from which no furtherincrease in dispersion was observed. In contrast, mild heating andstirring of the IVV drug substance (30 mins at 37° C. with stirring 600rpm) did not alter the level of dispersed material.

The intensity at which energy was transferred into IVV drug substancewas explored by adjusting the sonication amplitude (0-100%) while theexposure time remained constant at 0.70 s/ml for all samples. ODTresults indicated a predictable trend as previously seen with respect tothe amount of energy transferred, where the extent of dispersion in IVVdrug substance increased with sonication amplitude when exposure timewas kept consistent (FIG. 3). An optimal rate of sonication energytransfer was achieved at 80% amplitude, beyond which there is no furtherelevation in the level of agglomerate dissociation. These resultssuggest 80% is an optimal amplitude (i.e. rate of energy transfer) fordissociating agglomerates.

Feasibility of Sonication for Dispersing IVV Drug Matrix Material

To be acceptable inactivated vaccines require consistent qualityattributes. That is, if a method has been shown to disperse agglomeratedmaterial it is important that the material retains this characteristic.

A study was conducted over 24 weeks to assess the feasibility ofsonication as a method to disperse agglomerated material in IVV DrugMatrix of H3N2 A/Victoria/210/2009, both in the presence and absence ofa detergent, Polysorbate 80 (PS80). To monitor various characteristicsof the samples a multitude of tests were employed, including ODT and DLSfor agglomeration assessment, single radial immunodiffusion (SRID) forantigenicity and electron microscopy (EM) for morphological imaging.

Agglomeration Behavior by ODT and DLS

The agglomeration characteristics of the untreated, sonicated andPS80-treated IVV Drug Matrix were evaluated by the ODT assay and DLS.ODT analysis demonstrated that following sonication (at least 200Joules/mL), the level of dispersed material in IVV Drug Matrix reached80% compared to 40% for the non-sonicated material (at time 0) (FIG. 4).This level of dispersion remained consistent over 24 weeks at 4° C.indicating an irreversibly dispersed state (FIG. 4). Further there wasno increase in dispersion or further agglomeration of the control(untreated) material over this time. The addition of a detergent (0.1%PS80) had no notable impact upon the level of agglomerates in thismaterial compared to the initial level. Hence, this indicated the levelof agglomeration of IVV Drug Matrix material is set following disruptionof detergent.

In conjunction with the ODT assay, samples were analyzed by DLS tofurther characterize agglomeration. For each sample, DLS measurements(n=5) generated an intensity particle size distribution (PSD), whichdemonstrates the relative intensity of light scattered by particles invarious size populations. Results for DLS revealed a close correlationwith those of ODT for all three samples (FIG. 5). For example, theuntreated and PS80-treated IVV Drug Matrix samples demonstratedmultimodal PSDs with peaks present at 60, 400 and 7000 nm thusindicating agglomerates within (FIGS. 5A and B). However, sonicationtreatment produced a mono-modal distribution with a single distinct peakpresent at 300 nm, suggesting a uniform and well dispersed sample thatwas devoid of agglomerates (FIG. 5C). At each time point of analysis,all samples generated reproducible PSDs and further remained unchangedover the course of the 24 week period.

Antigenicity by SRID and EIA

The level of antigenic material was assessed by SRID to determinewhether sonication or the addition of detergent affected the influenzaantigen. SRID analysis indicated that regardless of whether the materialwas sonicated or treated in the presence of a detergent, the level ofantigenic material remained at the same potency as that of the untreatedsample and was constant over time (Table 1, FIG. 6).

TABLE 1 SRID results of untreated, detergent (PS80)-treated andsonicated IVV Drug Matrix (H3N2; A/Victoria/210/2009) over time. Potency(μg HA/mL) Weeks 0 2 4 8 12 24 Untreated 1451.5 1860.0 1829.2 1760.31934.4 1759.3 Sonicated 1722.5 1657.5 1829.2 1559.1 1779.8 1670.8 PS801740.0 1710.6 1654.5 1578.8 1780.7 1749.8

Morphological Imaging by EM

Imaging by EM was used to examine the morphological appearance of thesamples at 0, 1, 2 and 6 month time-points (FIG. 7). Notable differenceswere observed between sonicated and control IVV Drug Matrix samples.Control/untreated IVV Drug Matrix (with and without PS80) containedsignificant amounts of agglomerates throughout the entire 6 month timecourse, as represented by the darker regions in the micrographs. Incontrast, sonicated IVV Drug Matrix contained fewer agglomerates thatwere reduced in size and the appearance of the material remainedconsistent for the period of time examined. These observations reflectedthe results obtained from both ODT and DLS analyses, in which sonicatedmaterial was evidently more dispersed than IVV Drug Matrix that wasuntreated or incorporated a detergent (PS80).

Applicability of Sonication to all Seasonal Strains of Influenza

While H3N2 displays the greatest level of agglomeration (FIG. 1)compared to other seasonal strains, it is essential to demonstrate thatthis method is able to disrupt agglomerates of all strains. Fourseasonal strains were examined for the level of agglomerates dispersed,pre- and post-sonication (at least 200 Joules/mL), over a period of sixmonths (FIG. 8). In all virus preparations examined, the application ofsonication increased the level of dispersed material and this materialremained dispersed over a six month period. The elevation in agglomeratedispersion upon sonication treatment appeared more pronounced for thetwo sub-strains of influenza A, H3N2 and H1N1, in comparison with thetwo influenza B viruses derived from the Yamagata and Victoria lineages.For example, the level of dispersed material increased by approximately60-100% for the two A strains compared with a 3-10% increase for the Bstrains.

The ODT results were further corroborated by the data obtained from DLSanalysis of all four influenza strains. Intensity PSDs derived from fivereplicate measurements for each sample at 0, 3 and 6 months are shown inFIG. 8. The untreated sample of A/Victoria/361/2011 depicted thepresence of agglomerates of various size populations due its multimodalprofile (FIG. 9A), whereas the PSDs for A/California/7/2009,B/Hubei-Wujiagang/158/2009 and B/Brisbane/60/2008 had more monomodalstructure, hence suggesting a more uniform population of particles (FIG.9 C, E, G respectively). Post sonication, all four strains exhibiteddistributions that were characteristic of well dispersed IVV Drug Matrixwhich did not contain any agglomerates (FIG. 9 B, D, F, H).

Stability and Batch Consistency of Sonicated IVV Drug Matrix

IVV Drug Matrix representing 4 vaccine candidate types/subtypes weresonicated to determine both consistency and stability of applying acontrolled level of energy (Joules/mL) to achieve a target level ofagglomerate dispersion (Table 2, FIG. 8). IVV drug substance for eachrepresentative strain was divided into 6 sub aliquots. Three of the 6aliquots were independently exposed to at least 200 Joules/mL ofsonication and stored for up to 6 months at 2-8° C. along with theirun-sonicated control groups. Samples were taken from all groups at 0, 1,3 and 6 months and analysed by ODT for the level of agglomeratespresent. A significant change in the level of dispersed agglomerates wasobserved in sonicated samples when compared to un-sonicated controls forall sub-lots within the Influenza A strain subtypes:A/California/07/2009 and A/Victoria/361/2011. There was greatconsistency amongst sub-lots sonicated independently for bothrepresentative Influenza A strains. At time 0 all three sub-lots of theH1N1 and H3N2 strains met the target % ODT (>80%) after sonication, with% CVs of 2.1% and 1.1% respectively, well within the 10% limit. Less ofa change in the level of agglomerate dispersion was observed in lotsrepresenting B strains due to the low content of agglomerates existingin control groups. Post sonication the B strain sub-lots displayed thesame level of batch to batch consistency as lots representing the Astrains with % CVs for the B/Hubei Wujiagang/158/2009 andB/Brisbane/60/2008 at time 0 of 1.2% and 0.7% respectively. Importantlysub-lots representing all 4 seasonal influenza strains maintained theirelevated level of % ODT and batch to batch consistency for the entire 6month time-course, suggesting agglomerate disruption by sonication ispermanent.

TABLE 2 Batches (n = 3) of IVV Drug Matrix representing 4 seasonalinfluenza strains pre and post sonication over a 6 month time courseA/California/07/2009, A/Victoria/361/2011, B/Hubei Wujiagang/158/2009and B/Brisbane/60/2008 Pre or Post Months Sonication 0 1 3 6A/California/07/09 Pre % DDT 60.4 59.4 58.2 62.2 (H1N1) Post % DDT 95.790.7 91.8 93.1 Post % CV 2.1% 3.0% 2.3% 1.5% A/Victoria/361/11 Pre % DDT41.9 43 43.4 46.2 (H3N2) Post % DDT 86.7 84.2 84.6 84.7 Post % CV 1.1%1.2% 2.9% 5.1% B/Hubei Pre % DDT 88.1 90.6 93.3 91.7 Wujiagang/158/09Post % DDT 98.6 97.3 95.4 96.9 (B YAM) Post % CV 1.2% 5.0% 1.9% 3.8%B/Brisbane/60/08 Pre % DDT 92.2 93.5 92.5 93.3 (B YAM) Post % DDT 95.496 96.5 98.3 Post % CV 0.7% 1.7% 2.0% 1.6%

Linear Relationship for the Number of Predicted Glycosylation Sites onHA and the Amount of Sonication Required.

The enveloped glycoprotein; hemagglutinin (HA) is the sialic acidreceptor-binding protein of the influenza virus which enables it to dockwith host cells and escape from digestion once engulfed into anendosome. The globular head region of the HA molecule contains N-linkedglycosylation sites which overlap with antigenic sites and are thoughtto be involved in the shielding of these antigenic sites from binding byantibodies and major histocompatibility complex (Skehel et al., 1984;Jackson et al., 1994). In addition, structural complexity of N-glycansis positively correlated with HA-receptor binding specificity (Tsuchiaet al., 2002). The number of N-linked glycosylation sites in theglobular head region of HA has increased during the evolution of H1N1and H3N2 human Influenza A virus (Suzuki. 2011). We have determined arelationship between the numbers of predicted glycosylation sites on theHA molecule of Influenza A and the level of agglomeration present. Thenumbers of glycosylation sites were predicted by calculating probabilityscores using an algorithm available on the NetNGlyc 1.0 Server(http://www.cbs.dtu.dk/services/NetNGlyc/). To produce a probabilityscore the HA protein sequence of the strain in question is entered intothe algorithms submission panel and submitted for analysis. The softwareproduces a table of predicted glycosylation sites within the enteredsequence which are scored with 1-3 plus (+) symbols depending on thestrength of probability. A predicted HA glycosylation site probabilityscore (pGly score) is defined as the sum of the plus symbols for a givenoutput sequence. We propose that H3N2 strains that have a pGly score of≥16 require sonication of ≥90 Joules/mL and H1N1 strains that have apGly score≥11 require sonication of ≥90 Joules/mL (wherein more than 50%of the material is not aggregated).

TABLE 3 relationship between the numbers of predicted glycosylationsites on the HA1 molecule of Influenza A and the level of agglomerationpresent % Predicted HA Glyc. STRAIN YEAR ODT Sites (Prob. Score.) H3N2A/Hiroshima/52/05 2005 62 14 A/Wisconsin/67/05 2005 86 16A/Brisbane/10/07 2007 44 17 A/Uruguay/716/07 2007 65 16A/Wisconsin/15/09 2009 55.4 16 A/Victoria/210/09 2009 42.8 17A/Victoria/361/11 2011 41.7 18 A/Texas/50/12 2012 33.3 18 A/SouthAustralia/55/14 2014 33.6 17 A/New Caledonia/71/14 2014 28.5 17 A/HongKong/4801/14 2014 32.0 19 A/Singapore/INFIMH-16-0019/2016 2016 18.0 19H1N1 A/California/07/09 2009 52.3 11 A/Singapore/GP1908/15 2015 24.7 12

Alternative Physical Methods of Disruption to Disperse IVV DrugSubstance

To assess the unique ability of sonication to disperse IVV Drug Matrix,other methods of physical disruption was examined. This includedlocalized heat (microwave for 1 and 10 s) and shear force (douncehomogenization using 25 and 100 strokes). In either case there was nonotable difference in dispersion compared to untreated material (Table4).

TABLE 4 ODT results for A/Victoria/361/2011 IVV drug substance aftermicrowave heating and shear force Dounce homogenization. Std dev % ODT(±) A/Victoria/361/2011 IVV drug substance; 38. 1 0.1 untreated(control) Microwave heating  1 second 37.2 0.3  10 seconds 39.0 0.5Dounce homogenization  25 strokes 40.5 1.5 100 strokes 39.6 0.3

CONCLUSIONS

The use of a direct sonication method has been found to effectivelydisperse agglomerates within IVV Drug Matrix. Influenza strains withinthe sub-type: H3N2 exhibit the highest levels of agglomeration.Optimization of the process indicated that the amount of energytransferred to IVV Drug Matrix and the rate at which transfer occurredwas essential in controlling the level of agglomerate dispersion.Increasing the rate of sonication (amplitude) and/or exposure time(seconds) resulted in an increase in the level of agglomeratedissociation which correlated with a linear trend. A plateau in thelevel of dispersion is observed after when the ODT reached 97% afterwhich little or no more agglomerates were dispersed (as measured byODT). The use of sonication as a method for dispersing agglomerates inIVV drug substance was evaluated over a time course of 24 weeks (6months), both in the presence and absence of a detergent (PS80). Severalcharacterization analytics including: ODT, DLS and EM revealed that thesonicated IVV Drug Matrix contained a significantly increased amount ofdispersed material compared with the untreated and detergent-treatedsamples. The amount of sonication required to reach a target level ofagglomerate dispersion could be predicted and was consistent betweenbatches. Further, the level of dispersion remained consistent over theentire duration of the feasibility study thus indicating a stable andpermanently agglomerate dispersed state. Immunological assessment bySRID confirmed that neither sonication nor detergent treatmentcompromised the antigenicity of the IVV Drug Matrix.

This work has strongly exemplified the value of sonication as a simple,practical and effective approach to improve the quality attributes ofinfluenza vaccines that contain highly agglomerated IVV Drug Matrix. Inaddition, this method has also been shown to be applicable to allseasonal strains of influenza. Following sonication treatment, increasedlevels of dispersed material was observed in IVV Drug Matrix of H3N2,H1N1 and two influenza B strains of the Yamagata and Victoria lineages,which was well maintained over a six month period.

As an alternative to the direct sonication method designed forlaboratory scale investigations, a continuous flow sonicationconfiguration was investigated for processing commercial volumes of IVVDrug Matrix. In brief, the Sonicator unit is powered by a high frequencygenerator combined with a convertor at 20 kHz; the connecting boosterhorn is encased by a flow-through processing vessel containing thesample that is constantly recirculated at a nominated flow rate.

To evaluate the scalability of the system, the influence of variousprocessing parameters, including: sonication intensity (amplitude) andproduct recirculation flow rate were investigated to determine theeffect on the efficiency of agglomerate dispersion within IVV drugsubstance. Using a constant recirculation flow rate through theSonicator of 120 ml/min and a fixed amplitude of 80%, a strong linearcorrelation was demonstrated between the batch volume (60, 500 and 1000ml) and sonication time (over 60 minutes) required to reach an ODTthreshold of 80% (R²=0.989). This trend was also observed betweensonication energy (Joules) and time (R²≥0.998), and hence betweensonication energy and batch volume of IVV Drug Matrix (R²=0.981, Table 5and FIGS. 10-12). This data suggests the sonication process outlined isscalable with respect to IVV drug substance batch size. Further, thedata suggests that a fixed energy input of at least 300 Joules/mL issufficient to dissociate agglomerates to an ODT level of ≥80%,regardless of volume in this system.

TABLE 5 Sonication time and energy input required to achieve an ODT ≥80%during recirculation of IVV Drug Matrix in the flow through Sonicatorfor various batch volumes (80% amplitude; 120 ml/min IVV Drug Matrixflow rate). IVV Drug Sonication time Energy Energy Matrix (sec) inputper mL ODT (ml) (sec) (Joules) (Joules/mL) (%) 60 150 18272 305 83 500660 89354 179 82 1000 1500 222953 223 83

We have determined a linear relationship between the number of predictedglycosylation sites on the Influenza A HA molecule and the degree ofagglomeration found in the IVV Drug Matrix of that strain (FIG. 13). Acorrelation co-efficient value (r value) was calculated for therelationship between predicted glycosylation sites and the degree ofagglomeration for IVV Drug Matrix of 12 H3N2 strains manufacturedbetween 2005 and 2017. The r value was 0.74 which suggests a moderatelystrong correlation for these two attributes. We propose that H3N2strains that have a pGly score of ≥16 require sonication of ≥90Joules/mL and H1N1 strains that have a pGly score≥11 require sonicationof ≥90 Joules/mL

REFERENCES

-   [1] CDC. Seasonal Influenza Vaccine Safety: A Summary for    Clinicians. 2011 [cited; Available from:    http://www.cdc.gov/flu/professionals/vaccination/vaccine_safety.htm-   [2] Fuminger IGS. Vaccine production. In: Nicholson K G, Webster R    G, Hay A J, editors. Textbook of influenza Oxford: Blackwell    Science, 1998: 324-32.-   Smith T L, Jennings R., Specificity and in vitro transfer of the    immunosuppressive effect of detergent-disrupted influenza virus    vaccine, Clin Exp Immunol. 1990 January; 79(1):87-94.-   Williams M S, Mayner R E, Daniel N J, Phelan M A, Rastogi S C,    Bozeman F M, et al. New developments in the measurement of the    hemagglutinin content of influenza virus vaccines by    single-radial-immunodiffusion. J Biol Stand. 1980; 8(4):289-96-   van de Donk H J, de Jong J C, van Olderen M F, Osterhaus A D.    Monoclonal antibodies for the control of influenza virusvaccines.    Developments in biological standardization. 1984; 57:251-5-   Bhatti A, Siddiqui, Y M., Micusan, VV. Highly sensitive fluorogenic    enzyme-linked immunosorbant assay: detection of staphylococcal    enterotoxin B 1. Journal of Microbiological methods. 1994; 19:179-87-   Skehel, J. J., Stevens, D. J., Daniels, R. S., Douglas, A. R.,    Knossow, M., Wilson, I. A., and Wiley, D. C. (1984) A carbohydrate    sidechain on hemagglutinins of Hong Kong influenza viruses inhibits    recognition by a monoclonal antibody. Annu. Rev. Biochem. 69,    531-569.-   Jackson, D. C., Drummer, H. E., Urge, L., Otvos, L. Jr., and    Brown, L. E. (1994) Glycosylation of a synthetic peptide    representing a T cell determininet of influenza virus hemagglutinin    results in loss of recognition by CD4+ T-cell clones. Virology 199,    422-430.-   Tsuchiya, E., Sugawara, K., Hongo, S., Matsuzaki, Y., Muraki, Y.,    Li, Z.-N., and Nakamura, K. (2002) Effect of addition of new    oligosaccharide chains to the globular head of influenza A/H2N2    virus hemagglutinin on the intracellular transport and biological    activities of the molecule. J. Gen. Virol. 83, 1137-1146.-   Suzuki, Y. (2011) Positive selection for gains of N-linked    glycosylation sites in hemagglutinin during evolution of H3N2 human    influenza A virus. Genes Genet. Syst. 86, 287-294.

1. A method of dispersing agglomerated material in a preparationcomprising influenza proteins, the method comprising subjecting thepreparation to sonication.
 2. The method according to claim 1, whereinthe preparation comprises influenza haemagglutinin.
 3. The methodaccording to claim 1, wherein the preparation comprises split influenzavirions.
 4. The method according to claim 3, wherein the preparation issubstantially free of detergent.
 5. The method according to claim 1,wherein the sonication is conducted for a time and at intensity suchthat at least 50% of agglomerates present in the preparation aredispersed.
 6. The method according to claim 1, wherein the sonication isconducted at a rate of 80% amplitude.
 7. The method according to claim1, wherein the sonication is conducted to transfer at least 90 Joules/mLof energy.
 8. The method according to claim 1, wherein H3N2 strains thathave a predicted HA glycosylation site probability score (pGly score)≥16require sonication of ≥90 Joules/mL and H1N1 strains that have a pGlyscore≥11 require sonication of ≥90 Joules/mL.
 9. A method of producingan influenza vaccine, the method comprising producing a preparationcomprising inactivated or split influenza virions and sonicating thepreparation.
 10. The method according to claim 9, wherein the vaccinecomprises at least 3 different influenza strains.
 11. The methodaccording to claim 9 wherein the vaccine is a monovalent vaccine. 12.The method according to claim 9, wherein the vaccine is a quadrivalentvaccine.
 13. The method according to claim 9, wherein the vaccinecomprises influenza A and influenza B.
 14. The method according to claim9, wherein the vaccine is substantially free of agglomerated material.15. The method according to claim 9, wherein the sonication is conductedat a rate of 80% amplitude.